JP2003057555A - Scanning laser microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To acquire all the spectral data of fluorescence from a specimen at a time and in a short time in a scanning laser microscope. SOLUTION: The scanning laser microscope acquires the data of a spectrum by converging a laser beam from a laser beam source 1 on the specimen of a observation target, irradiating the specimen with the laser beam, carrying out the movable scanning of the laser beam to the specimen, separating a light beam obtained from the specimen by means of a spectral means 15, and detecting the light beam by means of a detector 17. The detector 17 has an arrangement relation such that the detector 7 uses a one-dimensional photodetecting means in which a plurality of minute light receiving elements to generate an electric signal corresponding to an incidence light quantity are linearly arrayed so that the spectral output of the spectral means 14 enters within the range of the array of the minute light receiving elements of the one-dimensional photodetecting means with a relation predetermined in an incidence position and a wavelength band, and thus the data of the spectrum in unit of pixel is acquired.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a scanning laser microscope capable of obtaining spectral data by scanning a dyed specimen with a laser beam to disperse the light from the obtained specimen.

[0002]

2. Description of the Related Art In order to make cells and tissues easy to observe, staining is performed with a staining reagent. There are various types of dyeing reagents, and biological tissues are classified into reagents that are easily stained and those that are difficult to stain depending on the components. Utilizing this, a sample (specimen) is dyed with a desired reagent according to the purpose and subjected to microscope observation. And now, we take a step further and irradiate a sample stained with multiple types of fluorescent reagents with laser light, and collect the spectral data obtained by dispersing the fluorescence emission excited from the reagents by this laser light irradiation. , Used for analysis.

A scanning laser microscope is used for this purpose. In this method, a laser beam is collected through an objective lens, a sample is scanned by a scanning device, and the light from the sample is given to a spectroscope for spectroscopic analysis. In addition, the spectral information of the light from the laser irradiation position on the sample is obtained by obtaining the spectral data in synchronization with the scanning signal from the scanning device.

(Prior Art 1) As an example of a scanning laser microscope, there is a technology as disclosed in Japanese Patent Publication No. 9-502269. The technology disclosed herein disperses a fluorescent light beam by a spectrum resolving means such as a prism, narrows down a first spectral range on the one hand, and reflects at least a part of the spectral range that does not pass through the diaphragm on the other hand to form a second spectral range. The confocal scanning laser microscope has two optical paths and a photodetector is provided for each optical path.

FIG. 5 is a diagram showing the configuration of the confocal scanning laser microscope. The laser light flux 203 emitted from the laser light source 202 is guided to a dichroic mirror 208 via a direction changing mirror 204, a laser line filter 205, a lens 206, and a diaphragm 207, and is reflected there, and is then lens 209, XY scanning. It reaches the sample 213 via the optical system 210, the pupil projection lens 211, and the objective lens 212.

A light beam 214 composed of reflected light and fluorescent light from the sample 213 is an objective lens 212 and a pupil projection lens 21.
1, the XY scanning optical system 210, the lens 209, and the dichroic mirror 208. Then, of the light flux 214, the fluorescence component passes through the dichroic mirror 208 to become a fluorescence light flux 217, and of these, the light flux that has passed through the confocal diaphragm 215 is incident on the spectroscope 216.

FIG. 6 is a diagram showing the structure of the spectroscope 216. The selection device 225 comprises a spectral resolving means 227 for resolving the light beam 217 and, on the one hand, a first spectral range 22.
9 and 228 for reflecting at least a portion 230 of the spectral range which does not pass through the diaphragm. The photodetector 226 is the first photodetector 23 arranged in the optical path of the narrowed first spectral range 229.
1 and a second photodetector 232 installed in the optical path of the reflected spectral range.

The selection device 225 further comprises means 233 which is installed in the optical path of the reflected spectral range 230 and narrows down the second spectral range 234. The second photodetector 232 is installed in the optical path of the narrowed second spectral range 234.

(Prior Art 2) As another example, in Japanese Patent Laid-Open No. 2000-56244, both illumination light that is divided while being dispersed, such as reflected light and fluorescence, and light from an object are disclosed. Also disclosed is a scanning laser microscope having at least one selectively switchable micromirror array (DMD) for illumination and / or detection for wavelength selection of one.

The structure of the scanning laser microscope disclosed in this publication is as shown in FIG. That is, in this configuration, the laser light LB from the laser light source Ls is scanned by the scanning means Scn.
The sample Smp is scanned by XY scanning by moving the position intermittently, and the fluorescence excited by the laser beam LB is emitted from the sample Smp at the scanning position.

The (fluorescent) radiation emitted from the sample Smp is focused onto a confocal pinhole Ph located in a plane conjugate with the object. This pinhole Ph is also the opening of the spectroscopic device at the same time, and the spectroscope using the prism P separates the sample radiation into its spectral components by the dispersing action of the prism P. At the focal plane of the sample radiation is a one-dimensional DMD, where the sample spectrum is optically imaged.

The one-dimensional DMD consists of a number of individually switchable switching mirrors. Then, while the laser light LB from the laser light source Ls stays on the sample Smp, the mirrors are sequentially driven individually (and thus switched), so that the light that has been spectrally dispersed and arrived at that position. The detector Dtc
Send to. In this way the individual spectral components of the sample radiation are sequentially detected by the detector Dtc, and all spectral data of the (fluorescent) radiation emitted from the sample Smp is obtained.

As another method, a plurality of mirrors can be simultaneously driven to selectively guide light in a desired spectral range from a sample to a detector.

[0014]

Recently, in fluorescence observation, not only single staining but also multiple staining is frequently used. Of course, fluorescent staining is performed in order to make visible a specific object in cells or tissues. Therefore, in multiple staining observation, each stained site must be detected as a clear color difference, that is, a difference in fluorescence wavelength. Moreover, it is necessary to efficiently remove and detect the partial overlap (crossover portion) of the fluorescence wavelengths.

Further, in conducting research on cell functions, fluorescent probes for detecting intracellular calcium ion concentration have been widely used. This is because calcium ions have a close relationship with the activity status of cells. "INDO-1" as a fluorescent reagent for staining calcium ions
Is well known, but intracellular calcium ion concentration can be quantitatively measured by using this "INDO-1".

Sample stained with "Indo-1"
Emits fluorescence when irradiated with UV light (ultraviolet rays). Then, the generated fluorescence is detected in two different wavelength ranges (center wavelengths 405 [nm] and 480 [nm]), and the ratio of detected light amounts (fluorescence of center wavelength 405 [nm] / center wavelength 4
The calcium ion concentration can be measured by calculating and obtaining 80 [nm] fluorescence.

For a sample using a scanning laser microscope,
Laser light scanning is repeated, and the time change of intracellular calcium ion concentration is measured by the above calculation. In general, a dichroic mirror having an optical property of reflecting light in a specific wavelength band and transmitting the other light is split into two optical paths at 450 [nm], and one of them has a central wavelength of 405 [nm]. nm], a band width of about 20 [nm], and the other has a center wavelength of 480 [nm] and a band width of about 20 [nm]. Bandpass filters BPF1 and BPF2 are used to narrow the detection wavelength region.

However, the central wavelength of the fluorescence shifts depending on the state of the cell, for example, pH (pH; hydrogen ion concentration). For example, assuming that the fluorescence of Ca2 + max and Ca2 + min shifts their center wavelengths to O1 and O2 as shown in FIG. 8, in this case, the bandpass filter BPF
1, a deviation occurs between the center wavelengths BPF1o and BPF2o of BPF2 and the center wavelengths O1 and O2 of fluorescence Ca2 + max and Ca2 + min,
As a result, the amount of light that should be originally obtained cannot be obtained, so that not only the S / N (signal / noise) of the data is deteriorated, but also the calculation result represented by the light amount ratio is different from the value that should be originally obtained. Could be.

That is, the set of filters as described above in the prior art 1 is not always the best.

On the other hand, in the apparatus of Japanese Patent Publication No. 9-502269, the wavelength range of the fluorescence from the sample guided to each detector can be freely changed. Therefore, in solving the above problems in the prior art 1, , Considered effective. In fact, in multiple staining observation, for the purpose of efficiently removing and detecting the partial overlap (crossover portion) of the fluorescence wavelengths, the fluorescence from the sample is controlled by controlling the slit while checking the image by repeatedly scanning. Since the spectrum can be freely selected, it is far better than the method using an optical bandpass filter. However, it is desirable to reduce the laser scanning as much as possible because the sample is discolored by repeating the laser scanning.

However, in the example of measuring the intracellular calcium ion concentration, the state of the cell, for example, pH is unknown to the examiner, where the central wavelength of the fluorescence is, and the partial overlap of the fluorescent wavelengths ( Since there is no way to know in advance exactly what level the (crossover portion) is, there is a problem that it is not known whether the obtained result is the best or not.

That is, even if the change in the calcium ion concentration can be captured, the inspector does not know at all whether the result is the best one, and there is no guarantee that the best result is obtained. That is not the case.

If repeated tests can be conducted by trial and error, it is possible to bring the obtained results close to the optimum ones. However, in this type of test, once the data is acquired, basically, the sample must be exchanged (generally, the test is started by stimulating with a drug, but once the stimulus is given, the test is not performed. The second stimulation has no effect.) Therefore, it takes a lot of time and money to prepare the cells, and it must be said that it is difficult in reality.

As described above, the fundamental drawback of the prior art is that only a preset spectrum, that is, the average intensity of fluorescence passing through the slit can be used to form an image of the sample.

On the other hand, the technique disclosed in Japanese Unexamined Patent Publication No. 2000-56244 is used for obtaining the spectral data of fluorescence from the sample.
When collecting all spectroscopic data, it is necessary to drive the DMD mirror elements one by one to guide the spectroscopic spectrum of fluorescence from the sample to the photodetector. There is a drawback that it costs. Therefore, it becomes an unsuitable device for observing and measuring a phenomenon such as intracellular calcium ion concentration that changes rapidly. Therefore, in the prior art, it was not possible to observe and measure a phenomenon that changes rapidly such as intracellular calcium ion concentration. Therefore, urgent development of a technology to overcome this is urgently needed.

Therefore, the object of the present invention is that it is most suitable for performing a test or an imaging in which the fluorescence spectrum from the sample cannot be accurately known, as in the case of measuring the intracellular calcium ion concentration. It is an object of the present invention to provide a scanning laser microscope capable of reliably obtaining analysis results.

Another object of the present invention is to provide a scanning laser microscope capable of collecting the entire spectrum of light emitted from a dyed sample at one time.

Another object of the present invention is to provide a scanning laser capable of displaying an image reflected on a desired spectrum with a small number of scans in order to prevent fading of a sample in multiple staining observation. To provide a microscope.

Further, the object of the present invention is, in multiple staining observation, in order to prevent fading of the sample, a small number of scans, preferably one scan, results in partial overlap of fluorescence wavelengths (crossover). It is to provide a scanning laser microscope capable of efficiently removing (part) and displaying an image.

[0030]

In order to solve the above problems and achieve the object, the scanning laser microscope of the present invention is constructed as follows.

[1] A laser beam from a laser light source is focused and irradiated on a sample to be observed, the sample is moved and scanned, and the light obtained from the sample is dispersed by a spectroscopic means to be detected. By detecting with a vessel,
In the scanning laser microscope adapted to obtain spectrum data, the detector uses a one-dimensional photodetector means in which a plurality of micro light-receiving elements that generate electric signals corresponding to the amount of incident light are linearly arranged. The spectral output of the one-dimensional light detecting means is arranged in such a manner that the incident position and the wavelength region are incident on the array of minute light-receiving elements of the one-dimensional light detecting means with a predetermined relationship, whereby spectrum data is obtained for each pixel. To do.

The laser light from the laser light source is focused and irradiated on the sample to be observed, and the laser light is moved and scanned with respect to the sample, and the light obtained from the sample is dispersed by the spectroscopic means and detected by the detection means. By doing so, the spectral data for each point in the sample is obtained. The spectral data is obtained by using a spectroscopic means such as a prism, and by making the spectroscopic means disperse the light into the one-dimensional photodetecting means. The one-dimensional light detecting means has a structure in which a plurality of minute light receiving elements are linearly arranged (one-dimensionally), and the spectral output of the spectroscopic means is incident on the minute light receiving element arrangement range of the one-dimensional light detecting means. Since the position and the wavelength range are arranged so as to be incident in a corresponding relationship, the incident wavelength range is determined by the position of the micro light receiving element. Therefore, the light in the specific wavelength range of the spectrum is incident on the micro light receiving element at the specific position. Therefore, a signal corresponding to the amount of incident light is obtained from each micro light receiving element, and this is converted into digital data for each light receiving element, When the light receiving elements are collected in the order of arrangement, there is an effect that the wavelength components can be specified, that is, the data is collected as the data in which all the spectral distributions are known.

Moreover, since the laser light irradiating the sample moves and scans the sample, the above-mentioned data collected at each time is the data of the emission light at the irradiation point of the laser light on the sample, and the screen determined by the scanning range. It becomes the spectrum distribution data in units of pixels constituting the. Therefore, it is possible to collect as data that can grasp all the spectral distributions for each pixel.

Therefore, according to the present invention, a technique capable of acquiring all the spectral data of the fluorescence from the sample at once in a short time is established. Further, according to the present invention, when performing a test or imaging in which the fluorescence spectrum from the sample cannot be accurately known, as in the case of measuring intracellular calcium ion concentration, the optimum analysis result can be surely obtained. A scanning laser microscope capable of doing so can be provided.

Further, it is possible to provide a scanning laser microscope capable of collecting necessary fluorescence spectrum data with a small number of scans in order to prevent discoloration of a sample in multiple staining observation.

[2] In addition, in the configuration of [1],
Means for collecting electric signals obtained from each minute light receiving element of the detector and holding them as data, and using the held data, extract and add the data in the desired spectral range among these However, a means for generating an image is further provided.

According to this configuration, the data obtained by collecting the electric signals obtained from the respective minute light receiving elements of the detector are collected for the screen configuration, stored and stored, and the stored data is used to generate an image. However, since all the spectral distributions are collected for each pixel, it is possible to specify various spectral ranges as desired and display images with spectra in the specified ranges. After that, the optimum image can be found by generating various images using the data.

Therefore, according to the present invention, there is provided a scanning laser microscope capable of displaying an image reflected on a desired spectrum with a small number of scans in order to prevent discoloration of a sample in multiple staining observation. it can.

[3] In addition, in the configuration of [1],
A means for collecting electric signals obtained from each minute light receiving element of the detector and holding them as data, and using the held data, of these, a wavelength longer than the output laser wavelength of the laser light source is desired. While extracting the data of at least two specified spectral regions respectively and adding the extracted data in each spectral region,
It is provided with means for obtaining the ratio of the two and generating an image based on the ratio, and for outputting the generated image until the next image is generated, and means for displaying the generated generated image. Characterize.

In this structure, an image is displayed every time one scan is completed in the intermittent scan in which the scanning of one screen of the sample is performed again by the laser beam after a lapse of the scan of one screen. Therefore, the inspector can confirm how the image changes. Therefore, if the test is not working well, you can stop the test there and save time.

In addition, the present invention may include the following aspects.

(1) In the scanning laser microscope of the present invention, the laser beam is condensed through the objective lens, the sample is scanned by the scanning device, the light from the sample is projected on the spectroscope, and the scanning is performed. A scanning laser microscope capable of obtaining spectral data corresponding to a predetermined spectral band in synchronism with a scanning signal from the device, wherein in the spectral data, only data in a desired spectral region is extracted and calculated, Based on the result of this calculation, an image is displayed after the end of scanning.

(2) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) above, and the data calculation is performed again by changing the desired spectral range, and the calculation result is also obtained. The image is displayed again and the sequence is repeated.

(3) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) and (2) above.
In the spectral data, a plurality of desired spectral regions are designated, only the data in each spectral region is extracted and operated, and an image is displayed on at least one image plane based on the operation result. .

(4) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) and (2) above.
In the spectral data, a plurality of desired spectral regions are designated, only the data in each spectral region are extracted and calculated, and based on the calculation result, an image corresponding to the designated plural spectral regions is obtained. Each is displayed on each image plane.

(5) The scanning laser microscope of the present invention is the scanning laser microscope according to any one of (1) to (4) above,
The image display is performed based on the total value of data (light intensity data) in a desired spectral range.

(6) The scanning laser microscope of the present invention is the scanning laser microscope described in (3) above, in which two desired spectral regions are designated and only the data in each spectral region is extracted. While calculating the total value of each,
The ratio of these total values is calculated, and an image is displayed based on the calculation result.

(7) The scanning laser microscope of the present invention is the scanning laser microscope according to any one of (1) to (6) above.
The image display should be performed based on the total value of data (light intensity data) in the entire spectral range excluding the laser wavelength or the total value of data in the spectral range longer than this (light intensity data) excluding the laser wavelength. Is preset.

(8) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) to (6) above,
The image display should be performed based on the total value of data (light intensity data) in the entire spectral range excluding the laser wavelength or the total value of data (light intensity data) in the shorter spectral range excluding the laser wavelength. Is preset.

(9) The scanning laser microscope of the present invention is the scanning laser microscope described in (1) above, wherein the spectroscope includes a one-dimensional or two-dimensional CCD array.

(10) The scanning laser microscope of the present invention comprises:
In the scanning laser microscope according to the above (1), the sample scanning is intermittently performed a predetermined number of times for observation over time, and the image display is performed a predetermined number of times after the scanning.

[0052]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention makes it possible to obtain spectral data of fluorescence from a sample stained with a reagent over the entire spectral range in a short time. The analysis and image display can be performed by using the spectral data of a desired spectral region from the data. Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings.

(First Embodiment) FIG. 1 shows a first embodiment of the present invention.
It is a figure which shows the structure of the confocal scanning laser microscope concerning embodiment of this. In the figure, reference numeral 1 denotes a laser light source that oscillates and outputs a laser beam having a desired wavelength, and a measurement target is, for example,
When the intracellular calcium ion concentration labeled with Indo-1 is used, an argon laser light source having an oscillation line of 351 [nm] is used as the laser light source 1. Reference numeral 2 denotes a laser beam (laser beam) emitted from the laser light source 1.
Is.

Reference numeral 3 denotes a beam expander, which is an optical system for expanding the beam diameter of the laser beam 2 emitted from the laser light source 1 so as to approximately match the pupil diameter of the objective lens. Reference numeral 4 denotes a laser line filter, which is a filter that selectively transmits light having a desired wavelength set in advance. When the intracellular calcium ion concentration labeled with Indo-1 is to be measured, an optical filter that selectively transmits a wavelength of 351 [nm] is used. Reference numeral 5 denotes a dichroic mirror, which is a half mirror having an optical characteristic of reflecting light in a specific wavelength band and transmitting the other. When the measurement target has an intracellular calcium ion concentration, a mirror having optical characteristics that selectively reflects the wavelength of 351 [nm] and transmits the others is used.

Further, 6 is an XY scanning optical system, and 7
Is a pupil projection lens, 8 is an imaging lens, 9 is an objective lens, 1
0 is a sample. The sample 10 is dyed with a reagent according to the purpose. The objective lens 9 is a lens for adjusting the focus of the optical system with respect to the sample 10 so that the objective lens 9 can be moved back and forth close to the sample 10, and the imaging lens 8 and the pupil projection lens 7 have their optical axes of the objective lens 9. It is arranged to match the optical axis.

Further, 11 is a confocal lens, 12 is a confocal diaphragm, 13 is a collimating lens, and 15 is a prism. These confocal lens 11, confocal diaphragm 12 and collimating lens 13 collect light from the sample 10. They are arranged on the optical path leading to the prism 15 with their optical axes aligned.

The prism 15 is for separating the incident light of each wavelength.

The dichroic mirror 5 is arranged on the optical path for guiding the light from the sample 10 to the prism 15 and in the optical path between the confocal lens 11 and the XY scanning optical system 6 with its surface inclined. The laser light beam 1 guided from the laser light source 1 via the laser line filter 4 is reflected here to be guided to the sample 10 side, and the light from the sample 10 side is guided to the confocal lens 11 side. It is located at the intersection with the optical axis of.

The confocal lens 11 is a lens having a focal position conjugate with the focal position of the objective lens 9 with respect to the sample 10, and the confocal diaphragm 12 is a diaphragm arranged at the confocal position of the confocal lens 11. The confocal diaphragm 12 plays the role of a pinhole, and since the ideal pinhole diameter varies depending on the wavelength of the light to be extracted, it also has a function as a diaphragm so that it can be adjusted.

The fluorescence emitted from the sample 10 is focused on the confocal diaphragm 12 located in a plane conjugate with the observation object portion of the sample, so that the light passing through the confocal diaphragm 12 is spectroscopically determined. To the prism 15 of the spectroscope 14 to disperse the light.

The XY scanning optical system 6 is arranged in the optical path between the dichroic mirror 5 and the pupil projection lens 7 and can optically move the optical path within a predetermined range. Reference numeral 16 is a condenser lens, 17 is a one-dimensional CCD (solid-state image sensor in which pixels are arranged in a line), and the condenser lens 16 is a lens for collecting the light dispersed by the prism 15 into a predetermined range. The light is focused on the one-dimensional CCD 17. The one-dimensional CCD 17 is arranged at the focal position of the condenser lens 16.

As shown in FIG. 2, the one-dimensional CCD 17 is a line image sensor in which a plurality of light receiving cells corresponding to pixels are densely arranged in a line, and the light dispersed by the prism 15 is condensed by a condenser lens 16. 1-dimensional CCD 17
The light of a specific spectrum is made incident on a specific cell position and detected by being collected in the light receiving cell array range. Since the CCD 17 is configured to output pixel signals (luminance signals) due to the charges generated in the cells by the incidence of light as serial data in the order in which the cells are arranged at the time of reading, the position of the serial data of the pixel signals is Each of the configurations has a correspondence relationship that is a detection signal of a specific spectrum.

The prism 15, the condenser lens 16, and the one-dimensional CCD 17 compose the spectroscope 14. The laser light is used for XY scanning of the sample 10 by the XY scanning optical system 6, and the one-dimensional CCD after the spectroscopic analysis by the prism 15 of the fluorescence excited by the laser light at a certain moment.
The 17th output is a detection pixel signal that individually includes all the spectrum components included in the excitation fluorescence (or reflected light) at the position irradiated with the laser light.

Reference numeral 31 is a control device for driving and controlling the XY scanning optical system 6, and 30 is a signal processing device for outputting a signal corresponding to the incident light intensity detected by the one-dimensional CCD 17. , Is for converting into a digital signal and outputting in synchronization with the sampling signal of the control device 31.
Reference numeral 2 denotes a computer, which receives the data output from the signal processing device 30 and saves it as a file in a storage means, and also generates a spectral image using the saved files and displays a spectrum graph. Alternatively, it has a function of generating an image in the specified spectrum range by specifying the spectrum range. A monitor device 33 is an image display device for displaying an output screen of the computer 32.

It should be noted that the computer 32 is a GUI (Graphical User I) that is an interface that supports an image generation application and an inspector's operations and instructions easily
nterface; has an interface that operates a computer with a pointing device such as a mouse for graphical elements such as icons and windows displayed on the screen, and displays all spectral distributions collected for each image. Or specify at least one desired spectrum range individually from the displayed spectrum distribution, or extract data for the specified spectrum region from the storage means to generate and display an image. It has a function that is said to be.

Next, the operation of the present apparatus having such a configuration will be described. Now, let us consider a case where an attempt is made to measure intracellular calcium ion concentration. In this case, sample 10
"INDO-1" is used as a fluorescent reagent for staining.
In this case, an argon laser light source is used as the laser light source 1. The sample 10 stained with "INDO-1" is placed under the objective lens 9, and the objective lens 9 is focused.

After that, laser light is emitted from the laser light source 1. The laser light source 1 is an argon laser light source having an oscillation line (laser light) of 351 [nm], and the laser light flux 2 of 351 [nm] emitted from the laser light source 1 is
The beam expander 3 expands the beam diameter so as to substantially match the objective lens pupil diameter. Then, it is guided to the dichroic mirror 5 via the laser line filter 4.

The laser line filter 4 has 351 [nm].
Selectively transmits the oscillation line of. The laser beam having a wavelength of 351 [nm] that has passed through the laser line filter 4 is reflected by the dichroic mirror 5, passes through the XY scanning optical system 6, the pupil projection lens 7, the imaging lens 8, and the objective lens 9, and then the sample 10 Reach

Here, the laser beam focusing point on the sample 10 is determined by the objective lens 9, and the laser beam focusing position on the sample 10 is X- by the XY scanning optical system 6 for XY scanning the laser beam. Determined by the Y scan position.

The sample 10 is labeled with the fluorescent reagent "INDO-1", and when the sample 10 is irradiated with a laser beam having a wavelength of 351 [nm], the sample 10 which is the irradiation point is reflected. Fluorescence is generated by excitation with light or laser light. A light beam 20 composed of reflected light and fluorescence from the sample 10 returns to the dichroic mirror 5 via the objective lens 9, the imaging lens 8, the pupil projection lens 7, and the XY scanning optical system 6. Then, the light flux 20 passes through the dichroic mirror 5, where the light of the original laser light wavelength component is removed and only the light of the fluorescent component is transmitted, which further passes through the confocal lens 11 and the sample 10 Light from the irradiation point is focused and focused on the confocal diaphragm 12 side. Then, the confocal diaphragm 1 located at the focal point
2, the light from the position of the sample 10 which is the irradiation point is extracted, and the other light is removed by being removed from the diaphragm opening (pinhole) of the confocal diaphragm 12.
The light that has passed through the confocal diaphragm 12 is collimated (collimated) by a collimator lens 13 and enters a spectroscope 14.

The spectroscope 14 includes a prism 15 and a condenser lens 1.
6 and 1-dimensional CCD 17 confocal diaphragm 1
The light flux passing through 2 and collimated by the collimator lens 13 is spectrally decomposed by the prism 15, and is condensed on the one-dimensional CCD 17 through the condenser lens 16.
The one-dimensional CCD 17 is arranged at the focal position of the condenser lens 16, and moreover, a plurality of light receiving cells of a minute size are arranged linearly and densely, and the spectrum of the spectrum to be dispersed is The light is condensed by the condenser lens 16 so that the distribution range corresponds to the arrangement range of the light receiving cells in the one-dimensional CCD 17.

Therefore, there is a specific relationship between the arrangement position of each light receiving cell and the wavelength range of the spectrum to be dispersed. The wavelength of the spectrum obtained by the separation and the position of the light receiving cell for detecting the spectrum of the wavelength in the one-dimensional CCD 17 are determined.

Therefore, the light separated into each wavelength by being spectrally decomposed by the prism 15 is incident on the light receiving cell at the position corresponding to the spectroscopic optical path on the one-dimensionally arranged one-dimensional CCD 17. And one-dimensional CCD
In FIG. 17, the spectrum component of the output light at the laser light irradiation position at that time in the sample 10 is incident on the light receiving cell corresponding to the component, and as a result, the light receiving cell generates an electric charge corresponding to the amount of incident light.

The charges thus generated are sequentially read according to the arrangement order of the light receiving cells, and given to the signal processing device 30 as a light intensity signal continuous in the light receiving cell unit.
The signal processing device 30 converts the light intensity signal detected by the one-dimensional CCD 17 into a digital signal in synchronization with the sampling signal from the control device 31, and, for example, via a PCI bus which is a standard bus of a personal computer. Transfer to computer 32.

The computer 32 receives this and stores it in the storage means. The stored digital signal of each light receiving cell for one line is component-specific data (spectral distribution characteristic data) of all spectral components of the output light at the specific scanning position on the sample 10. Therefore, the component-specific data of all the spectral components of the output light at the specific scanning position on the sample 10 can be collected at once.

When the data collection at one point is completed,
The control device 31 moves the position of the laser beam 2 with respect to the sample 10 in order to drive and control the XY scanning optical system 6 in synchronization with the sampling signal generated by itself and acquire the spectrum data of the next pixel. Then, as described above, the light from that position is dispersed by the prism 15 and all the data (spectral distribution characteristic data) of the spectral components of the sample 10 at that position are collected.

The control device 31 drives and controls the XY scanning optical system 6 in synchronism with the sampling signal to move the position of the laser beam 2 with respect to the sample 10 and thereby the sample 10
The X-Y scanning is performed so as to cover each position of 1, and all the data of the spectral components of the sample 10 at each position are collected.

The one-dimensional CCD 17 described above is, for example,
It is a line image sensor with 256 light receiving cells, and the shortest wavelength is 350 [nm], and each element is about 1 [nm].
If the focal lengths of the prism 15 and the condenser lens 16 are optically set so as to correspond to
The dimensional CCD 17 has a structure capable of detecting a spectrum in the range of 350 [nm] to 605 [nm].

The signal processing device 30 is a one-dimensional CCD.
It is assumed that the output of 17 is digitized for each element with, for example, 8-bit resolution. Then, in this case, for each sampling signal of the control device 31 (that is, for one pixel (one pixel) on the image), 2
Data of 56 [Byte] (bytes) in capacity (data of spectral distribution characteristics) will be collected. The XY scanning optical system 6 covers 256 × 256 pixels, and if scanning is performed, a sampling signal for 256 × 256 times is output per screen, so one image (256 × 256 pixels) is output. ), The data capacity is 17
It will have a capacity of [MByte].

When one image of 256 × 256 pixel structure is to be acquired in 1 second, the transfer rate of data transferred from the signal processing device 30 to the computer 32 is 17
Since it is about [MByte / sec], it can be sufficiently transferred via the PCI bus mounted on a general computer.

Further, by installing a memory of 1 [GByte] in the computer 32, at least the image 50
It is possible to transfer the data of the spectral distribution characteristic for each pixel for one sheet to the computer and record it in the computer memory.

From the above, for the sample to be observed, the light intensity data for each component (data of the spectral distribution characteristic for each pixel) over the entire spectrum is included for each pixel for 50 seconds (50 images). ) Minutes of time observation data can be obtained.

Here, as shown below, the entire spectrum data string for each image pixel (Xi, Yj) n (where (Xi, Yj) is the pixel position, and n is the nth scanned image). I (K1) for each spectral component
n, I (K2) n, I (K3) n, ..., ..., I
(Km) n, for example, I (350) n, I (351)
, I, (605) n (n = 1 to 50, K1, K2, ...).
Indicates the wavelength, and if I (350) n, the incident light quantity value of the wavelength 350 [nm] of the nth image, and if I (351) n, the wavelength 351 [nm] of the nth image. Incident light intensity value, ...
Is shown).

The above is the data collection of spectral components in the scanning laser microscope of the present invention. Next, the image display method will be described.

After acquiring the data, the inspector selects a spectral range for image display. This is a computer 32
It is implemented by the software owned by.

The computer 32 has an application for image generation, a GUI which is an interface for supporting the operation and instructions of the inspector, and operating means. Then, a menu screen of these softwares is displayed on the monitor device 33 so that the inspector is instructed what process he wants to perform. If the inspector gives an instruction to generate an image of a desired spectral component from the collected spectrum data, the computer 32 receives this instruction.
Generates a designation screen of the spectrum range and sends it to the monitor device 33 for display. The inspector then indicates the desired spectral range.

For example, a distribution chart of spectral characteristics is graphically displayed from the collected spectrum data, and a desired spectrum range is designated on the displayed distribution chart. As a result, it is assumed that two spectral ranges are designated as indicated by the hatched areas with reference signs p and q in FIG. That is, one is from wavelength 390 [nm] to 420
Range (p) up to [nm], the other from 465 [nm] to 48
The range (q) is up to 5 [nm].

Upon receipt of this designation information, the computer 32
Is the spectrum data integrated value ΣI (K) of the specified range
n is calculated in pixel units. In the above example,

[Equation 1] Is calculated as. Then, the calculation is performed for each of the constituent pixels of the entire screen, and an image reflecting the result is generated.

An image corresponding to the spectrum of 390 [nm] to 420 [nm] is A, 465 [nm] to 485 [nm].
Let B be the image corresponding to the spectrum. Assuming that the fluorescence spectrum from the sample 10 moves to the short wavelength side as shown in FIG. 3B from the characteristics as shown in FIG. It can be seen that increases the brightness and image B darkens.

Then, in order to obtain the calcium ion concentration,

[Equation 2] However, α is an appropriate constant that can be set by the inspector. Is calculated.

For example, a sample (specimen) dyed with "INDO-1" is irradiated with UV light (ultraviolet light),
Fluorescence excited from the sample is detected in two different wavelength ranges (center wavelength 405 [nm] and 480 [nm]), and the ratio of detected light amounts (fluorescence of center wavelength 405 [nm] / center wavelength 480) is detected.
The calcium ion concentration can be measured by calculating and obtaining ([nm] fluorescence).

Therefore, the calcium ion concentration can be obtained by utilizing this fact and performing the calculation of [Equation 2] in the computer 32.

When such a calcium ion concentration is obtained for each pixel and the above calculation is completed for all the pixels on one screen, the computer 32 next produces an image in which the calcium ion concentration obtained for each pixel is reflected. To generate.

If the data for displaying an image representing the obtained calcium ion concentration is C, images A, B, and C are obtained for each image as shown in FIG. 1B. In the above example, A, B, and
An image of C will be obtained. These obtained images are displayed on the monitor device 33, and the inspector observes the displayed images.

Since 50 images have been obtained in the above example, the change in intracellular calcium ion concentration can be displayed as a moving image by sequentially displaying these 50 images.

In the scanning laser microscope apparatus shown in this embodiment, the data of all the original spectral components are held in the memory of the computer 32, so that the selected spectral range can be set to the computer 32. You can set it again and again by specifying it. Therefore, it is possible to obtain the best image showing the change in the calcium ion concentration by changing the range of the selected spectrum and comparing the states of the images.

As described above, in the device shown in this embodiment, the laser light from the laser light source is focused on the sample to be observed and irradiated, and the laser light is moved and scanned with respect to the sample to obtain the light obtained from the sample. In the scanning laser microscope, which is configured to obtain spectral data by dispersing the light with a spectroscopic means and detecting with a detector, the detector densely includes a plurality of minute light receiving elements that generate electric signals corresponding to the amount of incident light. Arrangement in which the linearly arranged one-dimensional light detecting means is used, and the spectral output of the spectroscopic means is incident on the array of minute light-receiving elements of the one-dimensional light detecting means such that its incident position and wavelength range have a predetermined relationship. It is a relationship.

Further, an optical system is provided for condensing the spectral output of the spectroscopic means so that the incident position and the wavelength range have a predetermined relationship in the array of minute light receiving elements of the one-dimensional photodetecting means.

Further, the signal processing means (the signal processing device 3 to obtain the data in the arrangement order of the minute light receiving elements by converting the output electric signal of each minute light receiving element of the one-dimensional light detecting means into a digital signal.
0) and holding means for holding the obtained data.

Further, the data held in the holding means is used to extract the data in the designated spectral range and generate the image with the spectral components in the designated range.

The laser light from the laser light source is focused and irradiated on the sample to be observed, and the laser light is moved and scanned with respect to the sample, and the light obtained from the sample is dispersed by the spectroscopic means and detected by the detector. By doing so, the spectral data for each point in the sample is obtained. The spectral data is obtained by using a spectroscopic means such as a prism, and by making the spectroscopic means disperse the light into the one-dimensional photodetecting means. The one-dimensional light detecting means has a structure in which a plurality of minute light receiving elements (light receiving cells) are densely arranged linearly (one-dimensionally), and the spectral output of the spectroscopic means is received by the minute light receiving means of the one-dimensional light detecting means. Since there is an arrangement relationship in which the incident position and the wavelength range are incident on the element array range in a relational manner,
The wavelength range of incident light is determined by the position of the minute light receiving element. Therefore, the light in the specific wavelength region of the spectrum is incident on the minute light receiving element at the specific position. Therefore, the data corresponding to the amount of incident light is obtained from each micro light-receiving element, converted into digital data individually for each light-receiving element, and the wavelength components can be specified by collecting the data in the order in which the light-receiving elements are arranged. Has the effect of being collected as.

Moreover, since the laser light irradiating the sample is moved so as to scan the sample in the XY direction, the above-mentioned data collected at each time is the data of the emission light at the irradiation point of the laser light on the sample, It becomes the spectral distribution data in units of pixels that form the screen defined by the XY scanning range. Therefore, it is possible to collect as data that can grasp all the spectral distributions for each pixel. This data is collected for the screen configuration, stored and stored, and an image is generated using this stored data. However, since it is collected as data that can grasp all spectral distributions for each pixel, various spectral ranges can be obtained as desired. An image with a spectrum of the designated range can be designated and displayed. Therefore, once the data is collected, various data can be used to generate the optimal image.

In fluorescent observation of a sample stained with a fluorescent reagent (fluorescent stained sample), not only single staining but also multiple staining is often used. In particular, fluorescent staining makes visible a specific object in cells or tissues. It is often done to Therefore, in multiple staining observation, each stained site must be detected as a clear color difference, that is, a difference in fluorescence wavelength. Moreover, it is necessary to remove and detect the partial overlap (crossover portion) of the fluorescence wavelengths.

In the present invention, all the spectral distributions can be collected for each pixel as data that can be grasped, and this data is collected and stored for the screen configuration, and an image in the desired spectral range is generated using this saved data. Since the data is saved, it is possible to repeatedly specify the spectrum range and specify the image as many times as necessary, so once the data is collected, the data can be used to generate various images. Now you will be able to find the optimal image.

Further, a plurality of ranges of spectral regions can be designated at the same time. Further, the data of the spectral components in the designated spectral regions are added to generate an image by the spectral components of the designated regions, and the image is further utilized. It is possible to obtain the ion concentration etc. by calculation, and the image of the obtained ion concentration can be displayed or combined with other images to be displayed, so that the inspector can be provided with detailed information about the test results. There is also an effect.

In the above example, a two-dimensional image is targeted, but it goes without saying that line scanning (one-dimensional) may be used. In this case, the spatial axis of scanning is taken in the horizontal direction of the image, and the time (or the number of times of scanning) is taken in the vertical axis. Further, although an example in which a prism is used for spectroscopy is shown, a diffraction grating may be used, and any means capable of performing spectroscopy can be used.

The above is the method in which the data of the spectral distribution of each pixel is collected and held, the desired spectral region is defined using the held data, and the image based on the component of the region is obtained. It is possible to grasp the situation while carrying out the procedure, and to judge whether the test is continued or discontinued depending on the situation, and if the test does not seem to be working well, you can take measures such as stopping the test there. Then, an embodiment in which time is not wasted will be described as a second embodiment.

(Second Embodiment) In this embodiment, at the time of collecting spectrum data, a total value of data in two spectrum regions longer than this excluding the output laser wavelength component of the laser light source is calculated in advance, and The ratio is calculated for each pixel, and the image is displayed based on the results of these calculations, and the image display is updated each time one screen of laser scanning for the sample is completed, and the progress is sequentially grasped. It enables you to do it.

FIG. 4A is a diagram showing the configuration of a confocal scanning laser microscope according to the second embodiment of the present invention. Also in this embodiment, the basic configuration may be the same as that described in the first embodiment. However, in the second embodiment, a helium-cadmium laser light source having an oscillation line of 442 [nm] is used as the laser light source 1, X-
The Y-scanning optical system 6 covers 512 × 512 pixels, and the one-dimensional CCD 17 has a scanning point.
A line sensor equivalent to 24 elements, with the shortest wavelength of 430
[nm], the focal lengths of the prism 15 and the condenser lens 16 are optically set to correspond to about 0.25 [nm] per pixel, and the one-dimensional CCD 17 is 400 [nm].
To 685 [nm] from the point where the computer 32 can detect the laser wavelength (442
[nm]) component is excluded, the total value of data in two spectral regions longer than this is obtained, the ratio for each pixel is calculated, an image is generated based on these calculation results, and the monitor device 33 It is set to display images, and X
-It differs from the first embodiment in that the image display on the monitor device 33 is updated every time the Y-scanning optical system 6 finishes scanning the laser beam in the pixel range for one screen. still,
Reference numeral 34 is a mass storage device such as a hard disk connected to the computer 32.

The operation of the apparatus having the configuration disclosed in this embodiment will be described. Here, the sample 10 to be observed is assumed to be a cell in which two types of fluorescent proteins (for example, two types of fluorescent proteins of CFP and YFP) are gene-expressed.

The laser light flux 2 emitted from the helium cadmium laser light source 1 having an oscillation line of 442 [nm] is
The beam expander 3 expands the beam diameter so as to substantially match the pupil diameter of the objective lens and guides it to the laser line filter 4. The laser line filter 4 selectively transmits the oscillation line of 442 [nm].

442 transmitted through the laser line filter 4
A laser beam having a wavelength of [nm] is reflected by the dichroic mirror 5, reaches the sample 10 through the XY scanning optical system 6, the pupil projection lens 7, the imaging lens 8 and the objective lens 9.

Sample 10 is a cell in which two kinds of fluorescent proteins, CFP and YFP, are gene-expressed, and a change in intracellular calcium ion concentration causes a change in energy transfer from CFP to YFP. The intracellular calcium ion concentration can be measured by measuring the ratio of the amount of light.

The laser light reaching the sample 10 excites the fluorescent protein at the arrival position or generates reflected light.

Here, the laser beam focusing point on the sample 10 is determined by the objective lens 9, and the laser beam focusing position on the sample 10 is X- by the XY scanning optical system 6 for XY scanning the laser beam. Determined by the Y scan position.

The light beam 20 consisting of the reflected light from the sample 10 and the fluorescent light returns to the dichroic mirror 5 via the objective lens 9, the imaging lens 8, the pupil projection lens 7 and the XY scanning optical system 6. Then, the light beam 20 passes through the dichroic mirror 5 and the confocal lens 11, and enters the spectroscope 14 via the confocal diaphragm 12 and the collimator lens 13.

The spectroscope 14 includes a prism 15 and a condenser lens 1.
The light flux which is composed of 6 and one-dimensional CCD 17 and is transmitted through the dichroic mirror 5 is spectrally decomposed by the prism 15 and is condensed on the one-dimensional CCD 17 through the condenser lens 16. The one-dimensional CCD 17 is a condenser lens 1.
It is arranged at the focal position of 6.

The signal processing device 3 is synchronized with the sampling signal of the control device 31 for driving and controlling the XY scanning optical system 6.
0 converts the light intensity signal detected by the one-dimensional CCD 17 into a digital signal and sends it to the computer 3 via the PCI bus.
Transfer to 2.

The one-dimensional CCD 17 is, for example, a line sensor having a light receiving cell equivalent to 1024 elements, and the shortest wavelength 43
The focal lengths of the prism 15 and the condenser lens 16 are optically set so as to correspond to 0 [nm] and about 0.25 [nm] per pixel. Therefore, the light receiving cell 1024 element covers the spectral range from 400 [nm] to 685 [nm] in order. Then, each light receiving cell generates an electric signal corresponding to the intensity of the light of the wavelength component corresponding to itself. That is, the one-dimensional CCD 17 has four
The spectrum from 00 [nm] to 685 [nm] is configured to be detectable.

The signal processing device 30 processes the output of the one-dimensional CCD 17 with 8-bit resolution for each element and outputs it as data arranged in the order of arrangement.

Therefore, the data output from the signal processing device 30 is 1 for one sampling signal of the control device 31 (that is, for one pixel on the image).
It has a data capacity of [KByte] (kilobytes).

Since the range of XY scanning by the XY scanning optical system 6 is 512 × 512 pixels per image, the control device 31 outputs a sampling signal corresponding to this, so that the image 1 sheet (512 x 512
The data capacity is 268 [MByte] per pixel.

When acquiring one image every 60 seconds,
The transfer rate required from the signal processing device 30 to the computer 32 is determined by the PCI installed in a general computer.
It can be adequately handled via the bus. Further, by writing this data in the mass storage device 34 such as a hard disk connected to the computer 32 each time the laser scanning for one screen of the sample is completed, at least the image 50 is displayed.
It is possible to record data for one sheet. It suffices if the sum of the data transfer time from the signal processing device 30 to the computer 32 and the write time to the hard disk (mass storage device 34) is smaller than the laser scanning interval time for each screen.

In this embodiment, the processing in the computer 31 calculates the total value of data in two spectral regions longer than this, excluding the laser wavelength (442 [nm]) component in advance, and calculates the ratio for each pixel. Is set, and an image is generated based on the calculation result and an image is displayed, and the image display is updated every time the laser scanning is completed.

That is, two spectral ranges are designated in advance. One is from 465 [nm] to 495 [n
m] and the other is from 520 [nm] to 550 [nm].

Upon receipt of this designation information, the computer 32
Is the spectrum data integrated value ΣI (K) of the specified range
n is calculated in pixel units. In the above example,

[Equation 3] Is calculated as. Then, the calculation is performed for each of the constituent pixels of the entire screen, and an image reflecting the result is generated.

An image corresponding to the spectrum of 465 [nm] to 495 [nm] is A, 520 [nm] to 550 [nm].
Let B be the image corresponding to the spectrum. As the calcium ion concentration rises, the fluorescence spectrum from the sample undergoes energy transfer, so that the image B becomes brighter and the image A becomes darker.

Then, in order to obtain the calcium ion concentration,

[Equation 4] However, α is an appropriate constant that can be set by the inspector. Is calculated.

Then, the image based on the data for displaying the image showing the calcium ion concentration obtained by the calculation is C
Then, as shown in FIG. 4B, the images A, B, and C are obtained for each image, so that, for example, when the scanning for the image of n = 1 is completed, the image of n = 1 is obtained. Can be generated, so the generated image is displayed. Since these obtained images are stored in the mass storage device 34 and displayed on the monitor device 33, the inspector observes the displayed images.

When the laser scanning for the image of n = 2 is completed, the image of n = 2 is generated and the mass storage device 34
The image of n = 2 is displayed instead of the image of n = 1, and the image of n = 3 is generated at the stage when the laser scanning for the image of n = 3 is completed. The image is stored in the storage device 34, and the image of n = 3 is displayed instead of the image of n = 2, and so on. Monitor device 33 for generated image
Update display to. As a result, it is possible to observe the time course while grasping the status of the test.

Since the generated image is held in the mass storage device 34, the held predetermined number of sheets (50 sheets in the above example) after the laser scanning is performed a predetermined number of times (in the above example, 50 screens). By sequentially displaying the images in (1), the change in intracellular calcium ion concentration can be displayed as a moving image and observed over time.

That is, in this embodiment, the sample scan is intermittently performed a predetermined number of times for observation over time, and the image display is performed at the end of each laser scan or laser scan for obtaining the entire number of images. It was performed a predetermined number of times after all was completed.

Further, since the spectral distribution data for each pixel collected by laser scanning is stored, the spectral range to be selected can be reset and redisplayed any number of times, so that the calcium ion concentration can be changed. You can get the best image to represent.

Thus, what is shown in this embodiment is
By focusing the laser light from the laser light source on the sample to be observed and irradiating it and moving and scanning the laser light with respect to the sample, the light obtained from the sample is dispersed by the spectroscopic means and detected by the detector. , In the scanning laser microscope, which is configured to obtain the spectrum data, when the spectrum data is collected, the output laser wavelength component of the laser light source is excluded in advance, and the total value of the two spectrum regions longer than this is calculated, It is configured to calculate the ratio for each pixel and display an image based on these calculation results.The image display is updated each time the scanning of the sample by the laser light is completed, and the progress is sequentially updated. , So that it can be grasped.

Therefore, since it is possible to grasp the situation while conducting the test, it becomes possible to judge whether the test is continued or stopped depending on the situation. If the test does not seem to be successful, the test is stopped there. It is possible to deal with such things as the above, and it is possible to obtain an effect such that time is not wasted.

Therefore, in this embodiment, after the scanning for one screen is finished, there is a delay and the scanning for the one screen of the sample is again performed by the laser beam. Since it is displayed, the inspector can confirm the images of CFP and YFP and the change in calcium ion concentration during the test. Therefore, if the test does not seem to be successful, it is possible to take a measure such as stopping the test at that time, thereby avoiding waste of time.

In the above example, a helium-cadmium laser light source is used as a laser light source for observation by one-photon excitation, but an ultrashort pulse laser light source having an infrared wavelength such as a mode-locked titanium sapphire laser is used. Two
Photon excitation observation is performed, the total value of data in two spectral regions shorter than this is calculated, the ratio for each pixel is calculated, and the image display is set based on these calculation results, and scanning is completed. The image display of the calculation result may be updated every time. Here, the two-photon excitation (two-photon excitation method) is a method in which a fluorescent molecule is locally excited at a focal plane by irradiating a long-wavelength laser beam having a wavelength that is a multiple of the absorption wavelength with high density. It is also called a multiphoton excitation method, and is a method that can obtain a sectioning image equivalent to the confocal observation method without pinholes.

The method of acquiring data by two-photon excitation using such ultrashort pulse long-wavelength laser light has little phototoxicity to cells and enables observation for a longer time.

Although a one-dimensional CCD is used in this example, the light flux from the sample is condensed by the condenser lens 16,
If the diameter of the diffraction spot formed here is larger than the pixel size of the CCD, a two-dimensional CCD may be used. As a result, when the spot diameter is large, the light from the sample can be efficiently detected, and the data with good S / N can be acquired.

The present invention is not limited to the examples shown in the above-mentioned embodiments, but can be modified in various ways.

Further, in the present invention, the embodiments include inventions at various stages, and various inventions can be extracted by appropriately combining a plurality of disclosed constituent elements. For example, even if some constituent elements are deleted from all the constituent elements shown in the embodiment, at least one of the problems described in the section of the problem to be solved by the invention is
When one of them can be solved and at least one of the effects described in the section of the effect of the invention can be obtained, the configuration in which some of the constituent features in the embodiment are deleted can be realized as the invention.

[0142]

As described above, according to the present invention, the spectrum data is provided for each scanning pixel, the spectrum range for image display can be freely selected, and the designation of the spectrum range can be changed many times. Therefore, even when performing a test or imaging in which the fluorescence spectrum from the sample cannot be accurately known, such as measuring intracellular calcium ion concentration, a highly flexible scanning laser that can obtain the optimum analysis result. A microscope can be provided.

Further, in multiple staining observation, a scanning laser capable of efficiently removing a partial overlap (crossover portion) of fluorescent wavelengths with a small number of scans and displaying an image in order to prevent fading of a sample. A microscope can be provided.

[Brief description of drawings]

FIG. 1 is a diagram for explaining a first embodiment of the present invention.

FIG. 2 is a diagram for explaining the present invention and is a diagram for explaining a configuration example of a spectroscope in the scanning laser microscope of the present invention.

FIG. 3 is a diagram for explaining an example of an obtained spectrum, an example of specifying a spectrum range, and an example of transition states thereof.

FIG. 4 is a diagram for explaining a second embodiment of the present invention.

FIG. 5 is a diagram for explaining a conventional technique.

FIG. 6 is a diagram for explaining a conventional technique.

FIG. 7 is a diagram for explaining a conventional technique.

FIG. 8 is a diagram for explaining a conventional technique.

[Explanation of symbols]

1 ... Laser light source, 2 ... Laser luminous flux 3 ... Beam expander, 4 ... Laser line filter 5 ... Dichroic mirror, 6 ... XY scanning optical system 7 ... pupil projection lens, 8 ... imaging lens 9 ... Objective lens, 10 ... Sample 11 ... Confocal lens, 12 ... Confocal diaphragm 13 ... Collimating lens, 14 ... Spectroscope 15 ... Prism, 16 ... Condensing lens 17 ... One-dimensional CCD, 20 ... Luminous flux from sample 30 ... Signal processing device, 31 ... Control device 32 ... Computer, 33 ... Monitor device 34 ... Hard disk drive (HDD)

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G02B 21/16 G02B 21/16 F term (reference) 2G020 AA04 BA02 BA20 CA01 CB04 CB23 CB43 CC02 CC13 CC26 CC63 CD06 CD14 CD24 CD36 CD37 2G043 AA01 BA16 DA02 DA06 EA01 FA02 GA07 GB19 HA01 HA09 HA15 JA02 JA04 JA05 KA02 KA05 KA09 LA03 NA01 NA05 2H052 AA07 AA08 AA09 AC04 AC12 AC15 AC34 AD35 AF07 AF14 AF25

Claims (3)

[Claims] 1. A detector which focuses a laser beam from a laser light source on a sample to be observed and irradiates the sample, moves and scans the laser beam with respect to the sample, and disperses the light obtained from the sample by a spectroscopic means. In the scanning laser microscope, which is configured to obtain spectrum data by detecting with a detector, the detector is a one-dimensional light detecting means in which a plurality of minute light receiving elements that generate electric signals corresponding to the amount of incident light are linearly arranged. And the spectral output of the spectroscopic means is arranged in such a manner that the incident position and the wavelength range are incident on the array of minute light receiving elements of the one-dimensional photodetector with a predetermined relationship, whereby spectral data is obtained on a pixel-by-pixel basis. A scanning laser microscope characterized in that 2. A means for collecting electric signals obtained from each minute light receiving element of the detector and holding the electric signals as data, and using the held data, data in a desired designated spectral range 2. The scanning laser microscope according to claim 1, further comprising: a unit for extracting and adding and generating an image. 3. A means for collecting electric signals obtained from each minute light receiving element of the detector and holding the electric signals as data, and using the held data, a wavelength longer than the output laser wavelength of the laser light source. That is, the data in at least two types of spectral regions designated as desired are respectively extracted, and the extracted data in each spectral region is added, and the ratio between the two is calculated to generate an image based on the ratio, The scanning laser microscope according to claim 1, further comprising: a unit for outputting the generated image and a unit for displaying the output generated image until the image is generated.